CRYO-EM STRUCTURAL STUDIES OF ADENOVIRUS CELL ENTRY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 低温电子显微镜(Cryo-EM)结合三维图像重建技术和新开发的时间分辨方法，为研究病毒与宿主细胞的相互作用以及病毒进入细胞过程中发生的动态结构变化提供了一种强有力的方法。事实上，这些方法通常是大(1亿道尔顿)大分子复合体和具有高移动性受体结合位点的病毒/受体复合体的唯一可行的结构方法，就像对腺病毒的假设一样。这些拟议的研究将阐明腺病毒/受体结合在细胞表面、内化到包被坑以及病毒颗粒从内体逃逸的结构事件。具体目的包括对腺病毒颗粒与阻止细胞进入的单抗Fab片段进行成像，以定位五肽基病毒成分上的Arg-Gly-Asp整合素结合部位。此外，与内化受体的可溶性形式整合素avb3和avb5结合的病毒复合体的结构将被解决，以便评估在受体结合时可能发生的结构变化。我们还将研究病毒和病毒/受体复合体的构象变化，这是改变pH值以模拟细胞外环境(PH 8)和早期内体环境(pH 6.0-6.5)的结果。低pH处理后的快速冻结(毫秒内)允许捕获结构中间体。另一个目标涉及计算腺病毒温度敏感突变体TSL的图像重建，该突变体对于细胞进入是有缺陷的。将分析TSL突变体和野生型之间的结构差异，以分离生产腺病毒进入细胞所必需的元件。这些冷冻-EM结构研究将提供对腺病毒细胞进入过程的更多了解，腺病毒是一种很有前途的体内基因治疗载体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cryo-electron microscopy (cryo-EM) combined with three-dimensional image reconstruction techniques and newly developed time-resolved methods provide a powerful way to examine virus-host cell interactions as well as the dynamic structural changes that occur during viral cell entry. In fact these methods often represent the only feasible structural approach for large (100 million Dalton) macromolecular complexes and virus/receptor complexes with highly mobile receptor binding sites, as has been postulated for adenovirus. These proposed studies will elucidate the structural events during adenovirus/receptor binding at thle cell surface, intemalization into coatedpits, and escape ofthe virus particle from the endosome. Specific aims include imaging the adenovirus particle complexed with a Fab fragment of a monoclonal antibody that blocks cell entry in order to localize the Arg-Gly-Asp integrin binding site on the penton base viral component. In addition, the structure of the virus complex ed with a soluble form of the internalization receptor, the integrins avb3 and avb5, will be solved in order to evaluate possible structural changes that occur upon receptor binding. We will also examine conformational changes in the virus and virus/receptor complexes as a consequence of varying the pH value to mimic both the extracellular (pH 8) and early endosomal (pH 6.0 - 6.5) environments. Rapid freezing (within milliseconds) after low pH treatment allows the trapping of structural intermediates. A further objective involves calculating an image reconstruction of the adenovirus temperature sensitive mutant, tsl, which is defective for cell entry. The structural differences between the tsl mutant and wild type will be analyzed to isolate elements essential for productive adenoviral cell entry. These cryo-EM structural studies will provide a greater understanding of the cell entry process of adenovirus, a promising vector for in vivo gene therapy.